Belén Pérez

Expression Analysis of Phenylketonuria Mutations EFFECT ON FOLDING AND STABILITY OF THE PHENYLALANINE HYDROXYLASE PROTEIN

Phenylketonuria is an autosomal recessive human genetic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the present work we have used different expression systems to reveal folding defects of the PAH protein caused by phenylketonuria mutations L348V, S349L, and V388M. The amount of mutant proteins and/or the residual activity can be rescued by chaperonin co-overexpression in Escherichia coli or growth at low temperature in COS cells. Thermal stability profiles and degradation time courses of PAH expressed inE. coli show that the mutant proteins are less stable than the wild-type enzyme, also confirmed by pulse-chase experiments using a coupled in vitro transcription-translation system. Size exclusion chromatography shows altered oligomerization, partially corrected with chaperonins coexpression, except for the S349L mutant protein, which is recovered as inactive aggregates. PAH subunit interaction is affected in the S349L protein, as demonstrated in a mammalian two-hybrid assay. In conclusion, serine 349, located in the three-dimensional structure lining the active site and involved in the structural maintenance of the iron binding site, is essential for the structural stability and assembly and also for the catalytic properties of the PAH enzyme, whereas the L348V and V388M mutations affect the folding properties and stability of the protein. The experimental modulation of mutant residual activity provides a potential explanation for the existing inconsistencies in the genotype-phenotype correlations.

Overview of mutations in the PCCA and PCCB genes causing propionic acidemia.

Propionic acidemia is an inborn error of metabolism caused by a deficiency of propionyl‐CoA carboxylase, a heteropolymeric mitochondrial enzyme involved in the catabolism of branched chain amino acids, odd‐numbered chain length fatty acids, cholesterol, and other metabolites. The enzyme is composed of α and β subunits which are encoded by the PCCA and PCCB genes, respectively. Mutations in both genes can cause propionic acidemia. The identification of the responsible gene, previous to mutation analysis, can be performed by complementation assay or, in some instances, can be deduced from peculiarities relevant to either gene, including obtaining normal enzyme activity in the parents of many patients with PCCB mutations, observing combined absence of α and β subunits by Western blot of many PCCA patients, as well as conventional mRNA‐minus result of Northern blots for either gene or β subunit deficiency in PCCB patients. Mutations in both the PCCA and PCCB genes have been identified by sequencing either RT‐PCR products or amplified exonic fragments, the latter specifically for the PCCB gene for which the genomic structure is available. To date, 24 mutations in the PCCA gene and 29 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements and a variety of splicing defects. A greater heterogeneity is observed in the PCCA gene—no mutation is predominant in the populations studied—while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different spectrum of mutations, only sharing some involving CpG dinucleotides, probably as recurrent mutational events. Future analysis of the mutations identified, of their functional effect and their clinical relevance, will reveal potential genotype–phenotype correlations for this clinically heterogeneous disorder. Hum Mutat 14:275–282, 1999.

Maternal polymorphisms 677C-T and 1298A-C of MTHFR, and 66A-G MTRR genes : Is there any relationship between polymorphisms of the folate pathway, maternal homocysteine levels, and the risk for having a child with down syndrome?

This study was aimed at analyzing the effect of mutations in three non‐synonymous SNP genes (677Cu2009>u2009T and 1298Au2009>u2009C of the methylenetetrahydrofolate reductase (MTHFR) gene, and 66Au2009>u2009G in the MTRR gene) on total plasmatic homocysteine (Hcy), in 91 mothers of Down syndrome (DS) infants and 90 control mothers. The comparison of both groups of mothers is a new way to determine if those mutations and their interactions increase the risk for DS. Material came from the case‐control network of the Spanish Collaborative Study of Congenital Malformations (ECEMC).

Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5? or 3? cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of (14)C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B12 metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC‐affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B12. The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real‐time PCR and subsequent high‐resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin‐incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients. Hum Mutat 30:1–9, 2009.

Propionic acidemia: identification of twenty-four novel mutations in Europe and North America

Propionic acidemia is an inherited metabolic disease caused by the deficiency of the mitochondrial protein propionyl-CoA carboxylase (PCC), one of the four biotin-dependent enzymes. PCC is a multimeric protein composed of two different alpha- and beta-PCC subunits, nuclearly encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause the clinically heterogeneous disease propionic acidemia. In this work we describe the mutational analysis of PCCA and PCCB deficient patients from different European countries (Spain, Italy, Belgium, Croatia, and Austria) and from America (mainly USA). We report 24 novel PA mutations, nine affecting the PCCA gene and 15 affecting the PCCB gene. They include six missense mutations, one nonsense mutation, one point exonic mutation affecting splicing, seven splicing mutations affecting splice sequences, and nine short insertions or deletions, only two in-frame. We have found a highly heterogenous spectrum of PCCA mutations, most of the PCCA deficient patients are homozygous carrying a unique genotype. The PCCA mutational spectrum includes a high proportion of short insertions or deletions affecting one nucleotide. In the PCCA mutant alleles analyzed we have also found one single nucleotide change, a novel nonsynonymous SNP. On the other hand, the PCCB deficient patients carry a more reduced spectrum of mutations, 50% of them are missense. This work represents an extensive update of the mutational study of propionic acidemia providing important information about the worldwide distribution of PA mutations and representing another essential part in the study of the phenotype-genotype correlations for the prediction of the metabolic outcome and for the implementation of treatments tailored to each PA patient.

Human propionyl-CoA carboxylase β subunit gene : Exon-intron definition and mutation spectrum in spanish and Latin american propionic acidemia patients

Propionyl-CoA carboxylase (PCC) is a mitochondrial biotin-dependent enzyme composed of an equal number of alpha and beta subunits. Mutations in the PCCA (alpha subunit) or PCCB (beta subunit) gene can cause the inherited metabolic disease propionic acidemia (PA), which can be life threatening in the neonatal period. Lack of data on the genomic structure of PCCB has been a significant impediment to full characterization of PCCB mutant chromosomes. In this study, we describe the genomic organization of the coding sequence of the human PCCB gene and the characterization of mutations causing PA in a total of 29 unrelated patients-21 from Spain and 8 from Latin America. The implementation of long-distance PCR has allowed us to amplify the regions encompassing the exon/intron boundaries and all the exons. The gene consists of 15 exons of 57-183 bp in size. All splice sites are consistent with the gt/ag rule. The availability of the intron sequences flanking each exon has provided the basis for implementation of screening for mutations in the PCCB gene. A total of 56/58 mutant chromosomes studied have been defined, with a total of 16 different mutations detected. The mutation spectrum includes one insertion/deletion, two insertions, 10 missense mutations, one nonsense mutation, and two splicing defects. Thirteen of these mutations correspond to those not described yet in other populations. The mutation profile found in the chromosomes from the Latin American patients basically resembles that of the Spanish patients.

Methylmalonic acidaemia leads to increased production of reactive oxygen species and induction of apoptosis through the mitochondrial/caspase pathway.

Methylmalonic acidaemia (MMA) is a heterogeneous group of rare genetic metabolic disorders caused by defects related to intracellular cobalamin (vitamin B12) metabolism. Increasing evidence has emerged suggesting that free radical generation is involved in the pathophysiology of neurodegenerative diseases, including some inborn errors of metabolism. We have previously identified in MMA patients several differentially expressed proteins involved in oxidative stress [mitochondrial superoxide dismutase (MnSOD) and mitochondrial glycerophosphate dehydrogenase (mGPDH)] and apoptosis by a proteomic approach. We have now extensively evaluated various parameters related to oxidative stress and apoptosis in cultured fibroblasts from a spectrum of patients with methylmalonic acidaemia. Fibroblasts from several MMA patients showed a significant increase in intracellular reactive oxygen species (ROS) content and in MnSOD expression level with respect to controls, suggesting a cellular response to intrinsic ROS stress. Moreover, we have demonstrated, using siRNA, that mGPDH is an important ROS generator in MMA patients. Cells from patients with MMA had a higher rate of apoptosis than those of controls and there was evidence that this process primarily involves the mitochondrial/caspase‐dependent pathway. ROS level–phenotype correlation revealed that patients with severe neonatal cblB disorder had elevated intracellular ROS content. These findings support the possible role of oxidative stress in the pathophysiology of methylmalonic acidaemia. Copyright

Analysis of the effect of tetrahydrobiopterin on PAH gene expression in hepatoma cells

Tetrahydrobiopterin (BH4)‐responsive phenylalanine hydroxylase (PAH) deficiency is a recently recognized variant of phenylketonuria, with a probable multifactorial molecular basis. In this study we have investigated the effect of BH4 on PAH gene expression in human hepatoma. Our results show that increased BH4 levels result in an enhancement of PAH activity and PAH protein, due to longer turnover rates, while PAH mRNA levels remain unchanged. This was confirmed for mutant PAH proteins (A309V, V388M and Y414C) associated to in vivo BH4 responsiveness, validating previous studies. We can conclude that there is no effect of the cofactor on PAH gene transcription, probably being the chemical chaperone effect of BH4 stabilizing mutant PAH proteins the major underlying mechanism of the response.

Alkaptonuria in the Dominican Republic: identification of the founder AKU mutation and further evidence of mutation hot spots in the HGO gene

Alkaptonuria (AKU, MIM 203500), the first human disease to be recognised as a recessive trait and Archibald Garrods prototype “inborn error of metabolism”,1,2 is a rare disorder of the phenylalanine and tyrosine catabolic pathway caused by the deficiency of homogentisate dioxygenase (HGO, EC 1.13.11.5) activity.3 AKU patients are homozygous, or compound heterozygous, for loss of function mutations in HGO .4 As a consequence of this defect, AKU patients cannot convert homogentisate to maleylacetoacetate, which results in homogentisic aciduria, ochronosis, and arthritis.5 AKU shows remarkable allelic heterogeneity. More than 40 different AKU mutations have been identified in a total of fewer than 100 unrelated patients from many different countries. In addition to the AKU mutations, 19 polymorphisms have been encountered within the human HGO gene (for a complete description of the HGO mutations and polymorphisms see the AKU database (http://www.cib.csic.es/~akudb/index.htm)). The analysis of the haplotype association of polymorphisms in the AKU chromosomes has been very useful for the identification of the different AKU alleles and for tracing their migration during recent human history. In this regard, it has been shown that the three most widespread AKU mutation in Europe, M368V, V300G, and P230S (representing 20%, 5%, and 5% of European AKU chromosomes, respectively) are not recurrent mutations. Instead they are probably old mutations that were introduced into Europe with the founder populations and have spread throughout western Europe with the different migrations.6 Analysis of the HGO mutations and polymorphisms has also shown that the GGG sequence motif (or its reverse complement CCC) is a mutational hot spot in the HGO gene.7nnAKU has a very low prevalence (1:100 000-250 000) in most populations. However, in certain areas, such as the Dominican Republic and Slovakia, the incidence of alkaptonuria is unusually high.8,9 In …